Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Serotonin N-acetyl transferase (SNAT), is the key enzyme in the synthesis of melatonin, and it is regulated in several ways. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. Last year, we showed that melatonin production in the chick pineal is controlled in two ways: by an endogenous clock that changes SNAT gene expression and by cyclic AMP which acts posttranslationally to regulate protein levels, most likely by protection of the enzyme protein from degradation by proteosomal proteolysis. This year, in collaboration with Mark Rollag (USUHS) and Maribeth Eiden (NIMH) we undertook to explore the possibility that melanopsin (a novel photopigment present in chick pineal cells) is the unknown photopigment that mediates photoentrainment, the process by which light resets the endogenous clock. We have constructed retroviral vectors carrying sense and antisense versions of melanopsin and other genes for candidate photopigments. Preliminary experiments transducing GFP into the chick pineal cells have been successful, as have preliminary attempts to overexpress and underexpress melanopsin. We plan to compare the effects of specific perturbations of melanopsin and other photopigment candidates on photoentrainment. We are also attempting to identify the unknown signal transduction pathway from the photopigment to the clock. We have undertaken to determine whether the ERK, or related, pathways play a role in photoentrainment. PD 98059, a specific inhibitor of MAPK activation, induced light-like phase shifts in the melatonin rhythm. In contrast, SB 203580, a putatively specific inhibitor of p38 kinase activity, evoked dark-like phase shifts in the melatonin rhythm. Our preliminary experiments have succeeded in measuring the active and inactive forms of MAPK directly, by immunoblot. Changes in the amounts of inactive and active MAPK, p38, and JNK, if present, in response to light, drugs, and other perturbations that entrain the circadian clock in these cells, should help determine and characterize the role of these kinases in the mechanism of phototransduction and entrainment.